1. Field of the Disclosure
The present disclosure is related to methods of purifying viral vectors such as adeno-associated virus (AAV) and/or recombinant adeno-associated virus (rAAV). More particularly, the present disclosure is related to methods of purification of AAV and/or rAAV using continuous flow centrifugation in a non-ionic density gradient and, preferably, in a single purification step. The present disclosure is also related to density gradients and flow through buffers for such continuous flow centrifugation methods.
2. Description of Related Art
Viral vectors are used to deliver genetic material into host cells for various gene therapy applications. During such gene therapy applications, the delivery of the genetic materials into the host cells uses the viral vector to carry therapeutic genes into a human cell.
Viral vectors are formed by modifying one or more viruses to deliver a desired genetic material into the host cell. For example, some viral vectors are formed by replacing the disease causing genes in a virus with genes that provide a desired effect in the host cell. The replacing of genes in the virus can be performed so that the viral vector retains its ability to infect and replicate.
As a result, the formation of viral vectors has more recently been focused on viruses that are known to infect the target species without causing any known disease. Stated another way, the formation of viral vectors has more recently been focused on non-pathogenic viruses. Viruses that have been the focus of much study for use in the development of viral vectors are adeno-associated virus (AAV) and/or recombinant adeno-associated virus (rAAV). To that end, a number of gene therapy pharmaceuticals using AAV and/or rAAV are in various stages of development, clinical trial, and/or human trial.
Current purification technologies and techniques available can deliver viral vectors of the required purity through the use of multiple purification techniques. These typically employ analytical batch centrifugation technique (e.g., tube rotor centrifugation) and/or chromatography techniques. When the batch centrifugation method employs a tube rotor, multiple runs of this separation step are necessary to process an entire batch/lot of product. These separate runs are pooled together to provide a desired volume of purified viral vector. The batch centrifugation processes may be considered a bottle neck in research, development, manufacturing, scale up, product optimization, and other uses of viral vectors. Thus, batch centrifugation may not be considered to be rapid enough and/or to be cost effective enough for certain uses.
It is believed the present disclosure that there is a continuing need for methods of purifying AAV and/or rAAV consistently in larger volumes efficiently and at lower costs than previously possible.